The cell types that may be present in any fine-needle aspiration biopsy (FNAB) of breast include epithelial cells (EC), myoepithelial cells (MEC), bipolar stromal cells (BSC), vascular pericytes/endothelial cells (VPEC), and adipose cells (AC). The recognition of most of these benign cellular elements in aspirates of the breast is relatively straightforward, based on distinct cytomorphologic criteria. However, there is controversy regarding the recognition of MEC because BSC are often referred to as MEC by cytopathologists. It is important to identify MEC in breast aspirates, because their presence has been associated with benign epithelial proliferations. In this study we used immunocytochemical methods on archival cytology slides with antibodies specific for MEC, calponin, and smooth muscle myosin heavy chain (SMMHC), to determine the distribution of MEC in FNAB of the breast and to ascertain the distribution of MEC in in situ and invasive carcinomas. Fifteen benign FNABS of breast and corresponding tissue biopsies were obtained along with 10 malignant FNABS and corresponding excisional breast biopsies from 1989-1993. Calponin and SMMHC antibodies were used on archival alcohol-fixed Papanicolaou-stained direct smears as well as the corresponding tissue sections. The distribution and pattern of positive immunostaining with both antibodies were recorded on the benign elements and the carcinomas for both cytologic and histologic slides. Benign breast tissues demonstrated strong continuous immunostaining for calponin and SMMHC of MEC. The interlobular stromal cells as well as intralobular stromal cells showed no immunostaining with either antibody. In cytologic preparations, MEC staining with calponin and SMMHC appeared as spindle cells between epithelial cells or along the edges of the epithelial groups. The bipolar stromal cells did not stain with either antibody. The tissues with DCIS (ductal carcinoma in situ) often showed the presence of MEC with strong calponin immunostaining, but sometimes the immunostaining was discontinuous or entirely absent around markedly dilated ducts. The SMMHC antibody was invariably negative, with architectural DCIS in dilated ducts. Two cases of DCIS with prominent periductal fibrosis or inflammation were positive for calponin, but the periductal stromal cells were calponin- and SMMHC-negative. Invasive carcinoma was negative for both calponin and SMMHC, but areas of DCIS were often positive in a discontinuous pattern. In conclusion, 1) Benign cellular elements from breast tissue FNAB showed strong continuous decoration of MEC with both calponin and SMMHC. Vascular pericytes and vascular smooth muscle were positive for both antibodies, but these cells were not observed in the FNAB. Benign proliferative epithelium showed no local increase in MEC with either antibody. Bipolar stromal cells in tissue and smears did not stain for MEC antibodies. 2) BSC did not correspond morphologically to MEC, and were not decorated with calponin or SMMHC. 3) Calponin-positive MEC were commonly associated with in situ ductal lesions, although they may at times have been discontinuous or absent entirely. DCIS may be recognized in FNAB by the presence of calponin-positive MEC associated with tumor cell groups. 4) Invasive carcinomas were invariably negative for MEC with these antibodies.